Adrian Handforth

Harmaline-induced tremor as a potential preclinical screening method for essential tremor medications

No preclinical method to evaluate potential new medications for essential tremor (ET) is available currently. Although harmaline tremor is a well known animal model of ET, it has not found utility as a preclinical drug screen and has not been validated with anti‐ET medications. We measured harmaline tremor in rats (10 mg/kg s.c.) and mice (20 mg/kg s.c.) with a load sensor under the cage floor and performed spectral analysis on 20‐minute epochs. The motion power over the tremor frequency bandwidth (8–12 Hz in rats; 10–16 Hz in mice) was divided by the motion power over the full motion frequency range (0–15 Hz in rats; 0–34 Hz in mice). The use of these measures greatly reduced data variability, permitting experiments with small sample sizes. Three drugs that suppress ET (propranolol, ethanol, and octanol) all significantly suppressed harmaline‐induced tremor. We propose that, with this methodology, harmaline‐induced tremor may be useful as a preclinical method to identify potential medications for ET.

Carbenoxolone and mefloquine suppress tremor in the harmaline mouse model of essential tremor.

Excessive olivo‐cerebellar synchrony is implicated in essential tremor. Because synchrony in some networks is mediated by gap junctions, we examined whether the gap junction blockers heptanol, octanol, carbenoxolone, and mefloquine suppress tremor in the mouse harmaline model, and performed an open‐treatment clinical study of mefloquine for essential tremor. Digitized motion was used to quantify tremor in mice administered harmaline, 20 mg/kg s.c. In mice the broad‐spectrum gap junction blockers heptanol, octanol (350 mg/kg i.p. each), and carbenoxolone (20 mg/kg) suppressed harmaline tremor. Mefloquine (50 mg/kg), which blocks gap junctions containing connexin 36, robustly suppressed harmaline tremor. Glycyrrhizic acid (related to carbenoxolone) and chloroquine (related to mefloquine), which do not block gap junctions, failed to suppress harmaline tremor in mice. Clinically, tremor was assessed with standard rating scales, and subjects asked to take 62.5, 125, and 250 mg mefloquine weekly for 12 weeks at each dose. None of the four human subjects showed a meaningful tremor reduction with mefloquine, likely because clinical levels were below those required for efficacy. In view of recent genetic evidence, the anti‐tremor mechanism of these compounds is uncertain but may represent a novel therapeutic target, possibly involving gap junctions other than those containing connexin 36.

Pilot efficacy and tolerability: A randomized, placebo‐controlled trial of levetiracetam for essential tremor

The purpose of this pilot single‐site study was to assess efficacy and safety of levetiracetam for essential tremor, using a placebo‐controlled, double‐blind, randomized crossover design with an interim analysis planned after completion of the first 10 to 15 subjects. The study was designed to detect a mean 30% reduction in composite tremor score, comparable to that of primidone or propranolol, which can be demonstrated with 30 or fewer subjects. Each treatment arm included baseline tremor assessments, a 4‐week medication titration, 2 weeks of stable dose, and treatment tremor assessments. Levetiracetam was titrated to 3,000 mg/day or to a lower maximal tolerated dose. The median age was 72 years, with 28 years median tremor duration. There was no statistically significant difference in response between placebo and levetiracetam on any tremor rating scale or accelerometry measure. The 95% confidence interval for the true mean difference between placebo and levetiracetam treatments was +18.5 to −22.5%, which excludes the minimum 30% drop required to consider levetiracetam clinically effective to a degree comparable to primidone or propranolol. Whether levetiracetam has lesser‐degree antitremor efficacy was not addressed in this pilot study.

Inhibition by dopamine agonists of dopamine accumulation following γ-hydroxybutyrate treatment

Abstract The increase in the dopamine (DA) concentration of rat (whole) brain induced by γ-hydroxybutyrate was inhibited by the reputed dopamine agonists apomorphine, apocodeine, 2-bromo-α-ergocryptine, piribedil, ergocornine and 5,6-dihydroxy-2-dimethylaminontetralin. The last three drugs raised DA levels in saline controls. Haloperidol, which decreased DA in controls slightly, prevented any agonist-induced rise in controls. However, haloperidol antagonized only apomorphine and piribedil in regard to the inhibition of the γ-hydroxybutyrate-induced rise in DA; this neuroleptic did not affect the inhibition by the other agonists. It is concluded that these data provide evidence for local receptor control of DA synthesis and that the DA agonists do not act through a common mechanism.

T-type calcium channel antagonists suppress tremor in two mouse models of essential tremor.

Essential tremor is a common disorder that lacks molecular targets for therapeutic development. T-type calcium channel activation has been postulated to underlie rhythmicity in the olivo-cerebellar system that is implicated in essential tremor. We therefore tested whether compounds that antagonize T-type calcium channel currents suppress tremor in two mouse models that possess an essential tremor-like pharmacological response profile. Tremor was measured using digitized spectral motion power analysis with harmaline-induced tremor and in the GABA(A) receptor α1 subunit-null model. Mice were given ethosuximide, zonisamide, the neuroactive steroid (3β,5α,17β)-17-hydroxyestrane-3-carbonitrile (ECN), the 3,4-dihydroquinazoline derivative KYS05064, the mibefradil derivative NNC 55-0396, or vehicle. In non-sedating doses, each compound reduced harmaline-induced tremor by at least 50% (range of maximal suppression: 53-81%), and in the GABA(A) α1-null model by at least 70% (range 70-93%). Because the T-type calcium channel Cav3.1 is the dominant subtype expressed in the inferior olive, we assessed the tremor response of Cav3.1-deficient mice to harmaline, and found that null and heterozygote mice exhibit as much tremor as wild-type mice. In addition, ECN and NNC 55-0396 suppressed harmaline tremor as well in Cav3.1-null mice as in wild-type mice. The finding that five T-type calcium antagonists suppress tremor in two animal tremor models suggests that T-type calcium channels may be an appropriate target for essential tremor therapy development. It is uncertain whether medications developed to block only the Cav3.1 subtype would exhibit efficacy.

Postseizure inhibition of kindled seizures by electroconvulsive shock

Abstract The inhibitory postseizure effect of electroconvulsive shock (ECS) and of kindled seizures on kindled seizures was assessed. Rats were kindled by daily amygdaloid stimulation. ECS delivered 22 or 24 h before each kindling stimulus markedly reduced the rate of kindling. In fully kindled rats multiple but not single ECS raised the threshold to kindled seizures 24 and 48 h after the last ECS. The threshold returned to normal after a rest period. In a subsequent experiment the threshold of fully kindled seizures was measured once daily for 8 consecutive days. The threshold was found to increase to a plateau, then return to normal after a 15-day rest. Multiple seizures are more effective than single seizures in inducing the postseizure effect; the phenomenon is not seizure specific. It is postulated that multiple seizures induce a homeostatic inhibiting response.

C‐terminal mechano‐growth factor induces heme oxygenase‐1‐mediated neuroprotection of SH‐SY5Y cells via the protein kinase Cϵ/Nrf2 pathway

Recently, a variant of insulin‐like growth factor‐1, mechano‐growth factor (MGF), has been discovered whose 24‐amino‐acid carboxy end is protective in models of stroke, nerve injury, and amyotrophic lateral sclerosis, suggesting broad‐spectrum neuroprotective properties. Moreover, we recently demonstrated in vitro and in vivo that a modified protease‐resistant 24‐amino‐acid MGF derivative (MGF24) protects dopaminergic neurons from oxidative stress‐induced apoptosis via induction of the stress response protein heme oxygenase‐1. However, the underlying mechanism by which MGF24 up‐regulates heme oxygenase‐1 expression is unknown. In this study, we demonstrate that MGF24‐induced heme oxygenase‐1 up‐regulation is dependent on activation of protein kinase Cϵ and NF‐E2‐related factor‐2 (Nrf2). MGF24 induces nuclear translocation of Nrf2, and siRNA knockdown of Nrf2 or of heme oxygenase‐1 prevents MGF24‐induced heme oxygenase‐1 up‐regulation and neuroprotection of SH‐SY5Y cells against 6‐hydroxydopamine‐induced cell death. Pharmacological inhibition of ERK, p38 MAPK, PI3K/Akt, or PKC signaling revealed that only PKC inhibition by GF109203X prevents MGF24s ability to protect against 6‐hydroxydopamine‐induced cell death. GF109203X also prevented MGF24‐induced Nrf2 nuclear translocation and heme oxygenase‐1 up‐regulation. siRNA knockdown of protein kinase Cϵ blocks MGF24‐induced Nfr2 nuclear translocation, heme oxygenase‐1 expression, and neuroprotection. Taken together, these results demonstrate that PKC activity is needed for MGF24s activation of Nrf2, which in turn increases heme oxygenase‐1 expression, a critical event in mediating MGF24s neuroprotection against 6‐hydroxydopamine‐induced apoptosis. Published 2011 Wiley‐Liss, Inc.

Zonisamide for Essential Tremor : An Evaluator-Blinded Study

In this evaluator‐blinded open‐treatment trial, subjects with moderate/severe upper limb essential tremor were titrated to 300 mg/day zonisamide, or adjusted to a lesser dose if symptoms warranted, as monotherapy or as adjunct to stable antitremor medication, followed by a 12‐week extension phase. The primary efficacy outcome variables were blinded rater videotaped/drawing tremor score changes at the Treatment and Extension visits compared to Baseline, based on Fahn‐Tolosa‐Marin and Postural Tremor Scales. Subjects also rated Functional Disabilities. Primary outcomes showed reduced tremor scores at the Treatment (P < 0.00001, n = 25) and Extension (n = 16) visits, at mean doses of 252 and 225 mg/day, respectively. Subject ratings indicated 200 mg/day was superior to 100 mg/day, whereas 300 mg/day produced no additional benefit, but instead was associated with more adverse symptoms, most commonly somnolence, poor energy, imbalance, and altered taste. Future double‐blind placebo‐controlled trials are warranted.

C-terminal mechano growth factor protects dopamine neurons: a novel peptide that induces heme oxygenase-1.

To assess potential efficacy of mechano growth factor (MGF) for chronic neurodegenerative disorders, we studied whether MGF protects dopamine (DA) neurons subjected to neurotoxic stress. We show that a short 24-amino acid C-terminal peptide of MGF (MGF24) upregulates heme oxygenase-1 (HO-1) expression and protects SH-SY5Y cells against apoptosis and cell loss induced by three DA cell-specific neurotoxins: 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenylpyridinium (MPP(+)), and rotenone. MGF24 maintains the mitochondrial membrane potential and blocks the release of mitochondrial apoptotic-inducing factor into the cytoplasm induced by 6-OHDA, MPP(+), and rotenone. Chemical inhibition of HO-1 with zinc protoporphyrin-IX prevents neuroprotection by MGF24 against the three neurotoxins. MGF24 does not activate Akt signaling nor does Akt inhibition block MGF24 protection of SH-SY5Y cells. In 6-OHDA-lesioned rats, central or peripheral MGF24 administration protects against the development of contralateral forelimb under-utilization, reduces ipsilateral nigral DA cell body loss, and attenuates tyrosine hydroxylase fiber loss in the ipsilateral striatum, independent of IGF-1 receptor activation. Peripheral MGF24 administration upregulates HO-1 expression in striatal and midbrain tissue. This report is the first to demonstrate that a small peptide, MGF24, upregulates HO-1, an important cell defense mediator, and protects DA cells, suggesting new strategies for neuroprotection in Parkinsons disease.

The Mibefradil Derivative NNC55-0396, a Specific T-Type Calcium Channel Antagonist, Exhibits Less CYP3A4 Inhibition than Mibefradil

A novel mibefradil derivative, NNC55-0396, designed to be hydrolysis-resistant, was shown to be a selective T-type Ca2+ channel inhibitor without L-type Ca2+ channel efficacy. However, its effects on cytochromes P450 (P450s) have not previously been examined. We investigated the inhibitory effects of NNC55-0396 toward seven major recombinant human P450s—CYP3A4, CYP2D6, CYP1A2, CYP2C9, CYP2C8, CYPC19, and CYP2E1—and compared its effects with those of mibefradil and its hydrolyzed metabolite, Ro40-5966. Our results show that CYP3A4 and CYP2D6 are the two P450s most affected by mibefradil, Ro40-5966, and NNC55-0396. Mibefradil (IC50 = 33 ± 3 nM, Ki = 23 ± 0.5 nM) and Ro40-5966 (IC50 = 30 ± 7.8 nM, Ki = 21 ± 2.8 nM) have a 9- to 10-fold greater inhibitory activity toward recombinant CYP3A4 benzyloxy-4-trifluoromethylcoumarin-O-debenzylation activity than NNC55-0396 (IC50 = 300 ± 30 nM, Ki = 210 ± 6 nM). More dramatically, mibefradil (IC50 = 566 ± 71 nM, Ki = 202 ± 39 nM) shows 19-fold higher inhibition of CYP3A-associated testosterone 6β-hydroxylase activity in human liver microsomes compared with NNC55-0396 (IC50 = 11 ± 1.1 μM, Ki = 3.9 ± 0.4 μM). Loss of testosterone 6β-hydroxylase activity by recombinant CYP3A4 was shown to be time- and concentration-dependent with both compounds. However, NNC55-0396 (KI = 3.87 μM, Kinact = 0.061/min) is a much less potent mechanism-based inhibitor than mibefradil (KI = 83 nM, Kinact = 0.048/min). In contrast, NNC55-0396 (IC50 = 29 ± 1.2 nM, Ki = 2.8 ± 0.3 nM) and Ro40-5966 (IC50 = 46 ± 11 nM, Ki = 4.5 ± 0.02 nM) have a 3- to 4-fold greater inhibitory activity toward recombinant CYP2D6 than mibefradil (IC50 = 129 ± 21 nM, Ki = 12.7 ± 0.9 nM). Our results suggest that NNC55–0396 could be a more favorable T-type Ca2+ antagonist than its parent compound, mibefradil, which was withdrawn from the market because of strong inhibition of CYP3A4.